KIBRA repairs synaptic plasticity and promotes resilience to tauopathy-related memory loss

Synaptic plasticity is obstructed by pathogenic tau in the brain, representing a key mechanism that underlies memory loss in Alzheimer’s disease (AD) and related tauopathies. Here, we found that reduced levels of the memory-associated protein KIdney/BRAin (KIBRA) in the brain and increased KIBRA protein levels in cerebrospinal fluid are associated with cognitive impairment and pathological tau levels in disease. We next defined a mechanism for plasticity repair in vulnerable neurons using the C-terminus of the KIBRA protein (CT-KIBRA). We showed that CT-KIBRA restored plasticity and memory in transgenic mice expressing pathogenic human tau; however, CT-KIBRA did not alter tau levels or prevent tau-induced synapse loss. Instead, we found that CT-KIBRA stabilized the protein kinase Mζ (PKMζ) to maintain synaptic plasticity and memory despite tau-mediated pathogenesis. Thus, our results distinguished KIBRA both as a biomarker of synapse dysfunction and as the foundation for a synapse repair mechanism to reverse cognitive impairment in tauopathy.

The FUGW2 plasmid containing CT-KIBRA was co-transfected with Δ8.9 and VSV-G packaging plasmids in HEK293 cells using calcium phosphate transfection.The lentivirus was propagated in HEK293 cells at 37C and was collected twice over a 48-hour period.Collected media containing the lentivirus was purified using sucrose gradient ultracentrifugation and resuspended in sterile PBS.Lentivirus titer was estimated using a p24 Rapid Titer Kit (Takara Bio USA, Inc).
Mice were anesthetized by isoflurane inhalation, and a stereotax was used to position the lentivirus injection directly into the mouse hippocampus using the following coordinates from bregma: anterior-posterior: -2.0, medial-lateral ±1.5, dorsal-ventral -1.8.The lentivirus was injected into the hippocampus at a rate of 0.5 µL/min.Mice received analgesic treatment with buprenorphine with one dose at the start of the surgery and two additional doses within 24 h after surgery.

Electrophysiology
Extracellular field recordings were performed from acute horizontal brain slices.Recording electrodes were placed in the molecular layer of the dentate gyrus.Slices were placed in recording chambers perfused with oxygenated ACSF solution heated to 30°C.Recording electrodes (~3 megaohms resistance) were filled with ACSF and lowered 50 µm into the dorsal blade of the molecular layer of the dentate gyrus.A bipolar tungsten electrode (FHC) was positioned ~150 µm away from the recording electrode to stimulate the perforant pathway inputs to the dentate gyrus.
Stimulus pulses were elicited at an intensity range from 0.25 µA -25 µA every 30 s with a 0.5 ms stimulus duration using a Model 2100 Isolated Pulse Stimulate (A-M Systems) to acquire the maximal fEPSP slope.The stimulus intensity was adjusted to 30% of the maximal fEPSP slope to record the baseline for LTP recordings.LTP recordings were performed in the presence of 100 µM picrotoxin (Sigma).Baseline fEPSPs were recorded for 20 min.Following the baseline, the stimulus intensity was increased to 60% of the maximal fEPSP slope for the theta burst stimulation (TBS) only.TBS consisted of 10 theta bursts applied every 15 s and each theta burst contained 10 bursts (4 pulses, 100 Hz) every 200 ms.After TBS the stimulus intensity was returned to the same level as during the baseline LTP recordings and fEPSPs were recorded for an additional 60 min.The fEPSP slope was normalized to baseline LTP responses.Recordings were acquired with WinLTP software (version 1.11b, University of Bristol) using a Multiclamp 700B amplifier (Molecular Devices).Recordings and analyses were performed blind to genotype.

Behavioral Tests
For the object-context discrimination test, mice were habituated in the testing room while in their home cage for 30 min before the start of testing.During the sample phase, mice explored two similar, but distinct contexts in two different 10 min sessions that were 30 min apart.Context 1 contained two identical objects (X1 and X2) in a white box that was cleaned with 70% ethanol.
Context 2 contained a different set of objects (Y1 and Y2) in a white box with black and white checkered wallpaper that was cleaned with 1% acetic acid.The mice were returned to their home cages for 4 h after the sample phase.For the test phase, one of the X objects in Context 1 was replaced with an incongruent Y object, and one of the Y objects in Context 2 was replaced with an incongruent X object.Half of the mice, balanced by genotype and sex, were tested in Context 1 and the other half were tested in Context 2. The amount of time mice spent exploring the congruent and incongruent objects during a 10-min session was calculated.Videos of the sessions were manually scored by an experimenter who was blind to mouse genotype and treatment.
Y-maze arena was constructed with plexiglass having three arms of equal length (20 cm) at equal angles.Mice were placed in the center and allowed to freely explore the arena for 5 min and recorded by video (Noldus).Each mouse was manually scored for the sequence and number of arm entries in the 5-min period and the experimenter was blind to the genotype and treatment of each mouse.An arm entry was defined as having all four paws within one arm.The percentage of spontaneous alternations for each mouse was calculated as the number of alternations divided by the total possible number of alternations in the 5-min session.An alternation is defined as the mouse having entered all three arms in succession without revisiting a previously entered arm.
The Morris water maze used a pool with a diameter of 120 cm and filled with water at a temperature 22  1C made opaque by the addition of white tempera paint.Visual cues were placed around the pool.The experimenter was blind to the genotype and treatment of each mouse.Mice were pretrained for one day with 4 trials to find a submerged hidden square platform (14 cm x 14 cm) that was 1.5 cm below the water surface located in the center of a rectangular channel.The day after pretraining, each mouse performed 4 days of hidden platform training with 2 trials per day where they had 60 s to find and sit independently on the platform for 10 s.Upon completion of hidden platform training, the platform was removed from the pool and mice were tested in a single probe trial for 60 s on subsequent days.Following probe trials, all mice were tested in cued platform training.Each mouse was recorded and tracked during hidden platform training, probe trials, and cued platform training using EthoVision XT software (Noldus).
Proteolytic digestion.Aliquots of 150 µg protein for each tissue sample were subjected to lysis buffers containing 5% SDS and 50 mM triethylammonium bicarbonate (TEAB), at pH ~7.55.The samples were reduced in 20 mM dithiothreitol (DTT) in 50 mM TEAB for 10 minutes at 50⁰ C, subsequently cooled at room temperature for 10 minutes, and then alkylated with 40 mM iodoacetamide (IAA) in 50 mM TEAB for 30 minutes at room temperature in the dark.Samples were acidified yielding a final concentration of 1.2% phosphoric acid, resulting in a visible protein colloid.Subsequently, 90% methanol in 100 mM TEAB was added at a volume of 7 times the acidified lysate volume.Samples were vortexed until the protein colloid was thoroughly dissolved in the 90% methanol solution.The entire sample volume was spun through micro S-Trap columns (Protifi) collecting the flow-through in an Eppendorf tube (in 200 µL aliquots for 20 seconds at 4,000 x g), and importantly binding the samples to the S-Trap columns.Subsequently, the S-Trap columns were washed with 200 µL of 90% methanol in 100 mM TEAB (pH ~7.1) twice for 20 seconds each at 4,000 x g.S-Trap columns were placed into a clean elution tube and incubated for 1 hour at 47⁰ C with 125 µL of trypsin digestion buffer (in 50 mM TEAB, pH ~8) at a 1:25 ratio (protease:protein, wt:wt).The same mixture of trypsin digestion buffer was added again for an overnight incubation at 37⁰ C. Peptides were sequentially eluted from S-Trap micro spin columns with 50 mM TEAB, 0.5% formic acid (FA) in water, and 50% acetonitrile (ACN) in 0.5% FA.
Desalting.After centrifugal evaporation, samples were resuspended in 0.2% formic acid (FA) in water and desalted with Oasis 10-mg Sorbent Cartridges (Waters, Milford, MA).Samples were then subjected to an additional centrifugal evaporation and were finally re-suspended in 0.2% FA in water with a final concentration of 1 µg/µL.One microliter of indexed Retention Time Standard (iRT, Biognosys, Schlieren, Switzerland) was added to each sample.
Mass spectrometry system.Briefly, samples were analyzed by reverse-phase HPLC-ESI-MS/MS using an Eksigent Ultra Plus nano-LC 2D HPLC system (Dublin, CA) with a cHiPLC system (Eksigent) which was directly connected to a quadrupole time-of-flight (QqTOF) TripleTOF 6600 mass spectrometer (SCIEX, Concord, CAN).After injection, peptide mixtures were loaded onto a C18 pre-column chip (200 µm x 0.4 mm ChromXP C18-CL chip, 3 µm, 120 Å, SCIEX) and washed at 2 µl/min for 10 min with the loading solvent (H2O/0.1% formic acid) for desalting.Subsequently, peptides were transferred to the 75 µm x 15 cm ChromXP C18-CL chip, 3 µm, 120 Å, (SCIEX), and eluted at a flow rate of 300 nL/min with a 3 h gradient using aqueous and acetonitrile solvent buffers.Data-dependent acquisitions (for spectral library building): For peptide and protein identifications the mass spectrometer was operated in data-dependent acquisition (DDA) mode, where the 30 most abundant precursor ions from the survey MS1 scan (250 msec) were isolated at 1 m/z resolution for collision induced dissociation tandem mass spectrometry (CID-MS/MS, 100 msec per MS/MS, 'high sensitivity' product ion scan mode) using the Analyst 1.7 (build 96) software with a total cycle time of 3.3 sec as previously described (83).
The variable window width is adjusted according to the complexity of the typical MS1 ion current observed within a certain m/z range using a DIA 'variable window method' algorithm (more narrow windows were chosen in 'busy' m/z ranges, wide windows in m/z ranges with few eluting precursor ions).DIA acquisitions produce complex MS/MS spectra, which are a composite of all the analytes within each selected Q1 m/z window.The DIA cycle time of 3.2 sec included a 250 msec precursor ion scan followed by 45 msec accumulation time for each of the 64 variable SWATH segments.
Mass-spectrometric data processing, quantification and bioinformatics.Mass spectrometric data-dependent acquisitions (DDA) were analyzed using the database search engine ProteinPilot (SCIEX 5.0 revision 4769) using the Paragon algorithm (5.0.0.0,4767).Using these database search engine results a MS/MS spectral library was generated in Spectronaut 14.2.200619.47784(Biognosys).The DIA/SWATH data was processed for relative quantification comparing peptide peak areas from various different time points during the cell cycle.For the DIA/SWATH MS2 data sets quantification was based on XICs of 6-10 MS/MS fragment ions, typically y-and b-ions, matching to specific peptides present in the spectral libraries.Peptides were identified at Q< 0.01%, significantly changed proteins were accepted at a 5% FDR (q-value < 0.05).Differential expression analysis was performed using a paired t-test, and p-values were corrected for multiple testing, specifically applying group wise testing corrections using the Storey method (86).Gene Ontology (GO) term enrichment analyses were performed with Gene Set Enrichment Analysis (GSEA) (87,88) or ClueGO version 2.5.4 in Cytoscape version 3.7.1 (89,90).Enrichment analyses were applied from GO Cellular Compartments.

RNA extraction and quantification
Each well was plated with 0.5 x 10 6 HEK293 cells in a 12-well culture plate.The cells were transfected with HA-PKMζ or HA-PKMζ and CT-KIBRA-flag using Lipofectamine 2000.A day after transfection, mRNA from transfected HEK293 cells were extracted using a Total RNA Isolation Kit (Thermofisher).iScript cDNA Synthesis Kit (BioRad) was used to obtain cDNA from mRNA.PCR was performed on cDNA using the PKMζ primer pairs: PKMζ 'CTT ACA TTT CCT CAT CCC GGA AG' and 'TTC ACC ACT TTC ATG GCG TAA A'.Actin primer pairs 'ACA CCC TTT CTT GAC AAA ACC T', and 'CGC ATC TCA TAT TTG GAA TGA CT' were used for normalization.PCR products were analyzed using agarose gel electrophoresis and ImageJ software (NIH) was used for quantification of the intensity of the bands.
PKMζ (C*T*C*TTGGGAAGGCAT*G*A*C) and scrambled oligonucleotides (A*A*C*AATGGGTCGTCT*C*G*G) were generated (IDT) with phosphorothioate bonds (*).For chemical LTP experiments, rat neurons were preincubated with oligonucleotides (20 µM final) for 1 h before chemical LTP, and the oligonucleotides remained in the media for the duration of the chemical LTP experiment.